Project Summary/ Abstract Alphaviruses are mosquito-transmitted, positive-strand enveloped RNA viruses of the Togaviridae family that cause global disease in humans. At present, no antiviral agents or licensed vaccines exist for the treatment or prevention of any alphavirus infections. We recently used a genome-wide CRISPR/Cas9-based screen to identify the cell surface molecule Mxra8 as a novel entry receptor for multiple emerging alphaviruses that cause arthritis and musculoskeletal disease including chikungunya (CHIKV), Ross River (RRV), Mayaro, (MAYV), and O'nyong nyong (ONNV) viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced alphavirus infection of cells, and reciprocally, ectopic expression resulted in increased infection. Mxra8 bound directly to CHIKV particles and enhanced virus attachment and internalization into cells. We hypothesize that engagement of Mxra8 by different alphaviruses will explain how infection, tissue targeting, and disease pathogenesis occurs. The primary goals of this collaborative proposal between the Diamond, Fremont, and Smit laboratories are to define the precise mechanism(s) by which Mxra8 facilitates alphavirus entry into cells, to gain high- resolution structural insight as to how Mxra8 engages the alphavirus virion, and to determine the detailed role of Mxra8 in the pathogenesis of acute and chronic infection of multiple arthritogenic alphaviruses in vivo using novel murine models. The experiments in this proposal will define fundamental aspects of alphavirus biology that enhance our understanding of infection and cell tropism. This information may facilitate the development of small molecules or biologicals that disrupt Mxra8 interaction with alphavirus spike proteins, which could form the basis of future therapeutics that ameliorate disease of multiple emerging alphaviruses.